Method for achieving desired glial growth factor 2 plasma levels

ABSTRACT

The present invention relates to administering glial growth factor 2 (GGF2) to a patient in need thereof, to achieve serum levels of GGF2 within a desired therapeutic window determined based on the disease or disorder afflicting the patient. In a particular embodiment, the patient is suffering from a disease or disorder associated with reduced levels of myelination and the GGF2 is administered to promote myelination in the patient.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/002,608, filed Jan. 21, 2016, which is a continuation of U.S.application Ser. No. 13/853,386, filed on Mar. 29, 2013, now U.S. Pat.No. 9,272,015, issued Mar. 1, 2016, which is a continuation of U.S.application Ser. No. 12/380,760, filed on Mar. 2, 2009, now U.S. Pat.No. 8,410,050, and claims priority under 35 USC § 119(e) from U.S.Provisional Application Ser. No. 61/067,589, filed Feb. 29, 2008, thecontents of which are each herein incorporated by reference in theirentirety.

GOVERNMENT SUPPORT

The invention was made with U.S. Government Support under NationalInstitutes of Health (NIH) Grant No. RO1-NS45939-01. The United StatesGovernment has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to administering glial growth factor 2(GGF2) to a patient in need thereof, to achieve serum levels of GGF2within a desired therapeutic window determined based on the disease ordisorder afflicting the patient.

BACKGROUND

Neuregulins (NRGs) and NRG receptors comprise a growth factor-receptortyrosine kinase system for cell-cell signaling that is involved inorganogenesis in nerve, muscle, epithelia, and other tissues (Lemke,Mol. Cell. Neurosci. 7:247-262, 1996; Burden et al., Neuron 18:847-855,1997). The NRG family consists of three genes that encode numerousligands containing epidermal growth factor (EGF)-like, immunoglobulin(Ig), and other recognizable domains. Numerous secreted andmembrane-attached isoforms function as ligands in this signaling system.The receptors for NRGs are all members of the EGF receptor (EGFR)family, and include EGFR (or ErbB1), ErbB2, ErbB3, and ErbB4, also knownas HER1 through HER4, respectively, in humans (Meyer et al., Development124:3575-3586, 1997; Orr-Urtreger et al., Proc. Natl. Acad. Sci. USA 90:1867-71, 1993; Marchionni et al., Nature 362:312-8, 1993; Chen et al.,J. Comp. Neurol. 349:389-400, 1994; Corfas et al., Neuron 14:103-115,1995; Meyer et al., Proc. Natl. Acad. Sci. USA 91:1064-1068, 1994; andPinkas-Kramarski et al., Oncogene 15:2803-2815, 1997).

The three NRG genes, Nrg-1, Nrg-2, and Nrg-3, map to distinctchromosomal loci (Pinkas-Kramarski et al., Proc. Natl. Acad. Sci. USA91:9387-91, 1994; Carraway et al., Nature 387:512-516, 1997; Chang etal., Nature 387:509-511, 1997; and Zhang et al., Proc. Natl. Acad. Sci.USA 94:9562-9567, 1997), and collectively encode a diverse array of NRGproteins. The most thoroughly studied to date are the gene products ofNrg-1, which comprise a group of approximately 15 distinctstructurally-related isoforms (Lemke, Mol. Cell. Neurosci. 7:247-262,1996 and Peles and Yarden, BioEssays 15:815-824, 1993). Thefirst-identified isoforms of NRG-1 included Neu Differentiation Factor(NDF; Peles et al., Cell 69, 205-216, 1992 and Wen et al., Cell 69,559-572, 1992), Heregulin (HRG; Holmes et al., Science 256:1205-1210,1992), Acetylcholine Receptor Inducing Activity (ARIA; Falls et al.,Cell 72:801-815, 1993), and the glial growth factors GGF1, GGF2, andGGF3 (Marchionni et al. Nature 362:312-8, 1993).

The Nrg-2 gene was identified by homology cloning (Chang et al., Nature387:509-512, 1997; Carraway et al., Nature 387:512-516, 1997; andHigashiyama et al., J. Biochem. 122:675-680, 1997) and through genomicapproaches (Busfield et al., Mol. Cell. Biol. 17:4007-4014, 1997). NRG-2cDNAs are also known as Neural- and Thymus-Derived Activator of ErbBKinases (NTAK; Genbank Accession No. AB005060), Divergent of Neuregulin(Don-1), and Cerebellum-Derived Growth Factor (CDGF; PCT application WO97/09425). Experimental evidence shows that cells expressing ErbB4 orthe ErbB2/ErbB4 combination are likely to show a particularly robustresponse to NRG-2 (Pinkas-Kramarski et al., Mol. Cell. Biol.18:6090-6101, 1998). The Nrg-3 gene product (Zhang et al., supra) isalso known to bind and activate ErbB4 receptors (Hijazi et al., Int. J.Oncol. 13:1061-1067, 1998).

An EGF-like domain is present at the core of all forms of NRGs, and isrequired for binding and activating ErbB receptors. Deduced amino acidsequences of the EGF-like domains encoded in the three genes areapproximately 30-40% identical (pairwise comparisons). Moreover, thereappear to be at least two sub-forms of EGF-like domains in NRG-1 andNRG-2, which may confer different bioactivities and tissue-specificpotencies.

Cellular responses to NRGs are mediated through the NRG receptortyrosine kinases EGFR, ErbB2, ErbB3, and ErbB4 of the epidermal growthfactor receptor family (Busfield et al., 1997, Mol Cell Biol.17:4007-14; Carraway et al., 1997, Nature 387:512-6; Chang et al., 1997,Nature 387:509-12). High-affinity binding of all NRGs is mediatedprincipally via either ErbB3 or ErbB4 (Ferguson et al., 2000, EMBO J.19:4632-43). Binding of NRG ligands leads to dimerization with otherErbB subunits and transactivation by phosphorylation on specifictyrosine residues (Honegger et al., 1990, Mol Cell Biol. 10:4035-44;Lemmon and Schlessinger, 1994, Trends Biochem Sci. 19:459-63; Heldin,1995, Cell. 80:213-23; Hubbard et al., 1998, J Biol Chem. 273:11987-90).In certain experimental settings, nearly all combinations of ErbBreceptors appear to be capable of forming dimers in response to thebinding of NRG-1 isoforms. ErbB2, however, appears to be a preferreddimerization partner that may play an important role in stabilizing theligand-receptor complex.

GGF2 has been shown to promote proliferation, differentiation andprotection of Schwann cells (Goodearl et al., 1993, J Biol Chem.268:18095-102; Minghetti et al., 1996 J Neurosci Res. 43:684-93).Expression of NRG-1, ErbB2, and ErbB4 is also necessary fortrabeculation of the ventricular myocardium during mouse development(Meyer and Birchmeier 1995, Nature 378:386-90; Gassmann et al., 1995,Nature 378:390-4; Kramer et al., 1996, Proc Nati Acad Sci USA93:4833-8). GGF2 has also been shown to promote proliferation andprotection of cardiomyocyte cells (Zhao et al., 1998, J Biol Chem273:10261-10269). GGF2-mediated neuroprotection has also beendemonstrated in animal models of stroke, although parameters relating todosing remain undefined.

The present invention advances the use of GGF2 with respect totherapeutic applications by presenting guidance as to methods for GGF2administration that optimize therapeutic benefit, while limiting adverseeffects. The present invention defines target therapeutic windows forGGF2 serum concentration levels that are specified with respect toparticular disease conditions.

SUMMARY

The present invention relates to administering GGF2 to a patient in needthereof to achieve a serum plasma level of GGF2 within a targettherapeutic window determined to be effective in the treatment of adisease or disorder. In accordance with the present invention, GGF2 maybe administered in a pharmaceutical composition.

In accordance with the present invention, a method for avoidinginhibition of Schwann cell myelination following administration of glialgrowth factor 2 (GGF2) in a subject is presented, said methodcomprising: providing a subject in need of neuron myelination; providingGGF2 in a pharmaceutically acceptable carrier; administering the GGF2 tothe subject; and, determining that the amount of GGF2 is less than theamount that inhibits Schwann cell myelination.

In another embodiment, the present invention relates to a method forpromoting myelination in a patient afflicted with a disease or disorderassociated with reduced levels of myelination, the method comprising:selecting the patient afflicted with a disease or disorder associatedwith reduced levels of myelination; administering glial growth factor 2(GGF2) to the patient in an amount of about 500 ng of GGF2 per kg ofbody weight; whereby myelination is promoted.

In yet another embodiment, the present invention relates to a method forpromoting myelination in a patient afflicted with a disease or disorderassociated with reduced levels of myelination, the method comprising:selecting a patient afflicted with a disease or disorder associated withreduced levels of myelination; and, administering glial growth factor 2(GGF2) to the patient at an amount that achieves a plasma level of about0.01 nM GGF2.

In a further embodiment, the present invention relates to a method forbroadening the therapeutic dose range for GGF2 when GGF2 is used tofacilitate myelination, the method comprising: selecting a subject witha disease or disorder associated with reduced levels of myelination;administering GGF2 and a Mek1/Erk pathway inhibitor to the patient, and,whereby GGF2-mediated myelination is occurs at higher doses of GGF2 thanwould occur in the absence of administering the Mek1/Erk pathwayinhibitor.

In another embodiment, the present invention relates to a method fordetermining if an amount of GGF2 is a therapeutically effective amountfor promoting myelination, the method comprising: providing a subjectreceiving GGF2 therapy; and measuring c-Jun protein levels in thesubject, whereby an increase in c-Jun relative to baseline c-Jun levelsindicates that the amount of GGF2 is near a maximum threshold oftherapeutic efficacy for promoting myelination.

In a particular embodiment of the invention, GGF2 is administered to amammal using a dosing regimen directed to achieving a narrow targettherapeutic window of plasma GGF2 concentrations.

As indicated herein, GGF2 is known to be able to promote proliferation,differentiation and protection of Schwann cells. GGF2 has also beenshown to promote remyelination and reduce symptoms in animal models ofmultiple sclerosis including experimental autoimmune encephalomyelitis.Under some circumstances (e.g., at high concentrations of GGF2),however, GGF2 can prevent myelination of neurons co-cultured withSchwann cells.

The data presented herein demonstrate that GGF2 is indeed capable ofpromoting myelination of peripheral nerves but teach that precise dosingof GGF2 to a mammal in need thereof is required to achieve the desiredGGF2-mediated promoted myelination of peripheral nerves. As taughtherein, GGF2 is administered so as to be within a therapeutic window ofplasma GGF2 concentrations in order to promote myelination. In theabsence of the results presented herein, there is no appreciation of thenarrow therapeutic window of plasma GGF2 concentrations required topromote myelination in a mammal in need thereof.

The data presented herein also demonstrate that GGF2 is sufficient topromote myelination and rescue the myelination defect onCRD-Nrg1-deficient axons. At high concentrations, however, GGF2 inhibitsmyelination in an Erk-dependent manner. The present results demonstratethat GGF2 is capable of both promoting and inhibiting myelinationdepending on the concentration presented to the Schwann cells.

Accordingly, the present invention relates to the surprising discoverythat a hitherto unrealized positive correlation exists betweenGGF2-mediated PI3-kinase pathway activation and promotion of myelinationand a negative correlation exists between GGF2-mediated Mek1/Erk pathwayactivation and promotion of myelination. Alternatively stated, thepresent inventors discovered that administration of GGF2 can be finelytuned to promote myelination by assessing activation levels of thesepathways. In accordance with the present invention, a target therapeuticwindow for GGF2 with regard to promoting myelination in a subject is anamount of GGF2 that promotes PI3-kinase pathway activation (assayed, forexample, by detecting phosphorylated Akt) in the absence of detectableMek1/Erk pathway activation (assayed, for example, by detectingphosphorylated Erk).

The formulations and compositions of the present invention exhibit aspecific, desired release profile that maximizes the therapeutic effectwhile minimizing adverse side effects. The desired release profile maybe described in terms of the maximum plasma concentration of the drug oractive agent (C_(max)) and the plasma concentration of the drug oractive agent at a specific dosing interval (_(Ctau)). A ratio of C_(max)to C_(tau). (Cmax:C_(tau)) may be calculated from the observed C_(max)and C_(tau). A dosing interval (_(tau)) is the time since the lastadministration of the drug or active agent. In the present application,the dosing interval (_(tau)) may be, for example, twelve (12) hours, inwhich case C_(tau) is the concentration of the drug or active agent attwelve (12) hours from the last administration.

Additionally, the formulations and compositions of the present inventionexhibit a desired release profile that may be described in terms of themaximum plasma concentration of the drug or active agent at steady state(C_(maxSS)) and the minimum plasma concentration of the drug or activeagent at steady state (C_(min)ss). Steady state is observed when therate of administration (absorption) is equal to the rate of eliminationof the drug or active agent. A ratio of C_(max)ss to C_(min)ss(C_(max)ss: C_(min)ss) may be calculated from the observed C_(max)ss andC_(min)ss. In addition, the formulations and compositions of the presentinvention exhibit a desired release profile that may be described interms of the average maximum plasma concentration of the drug or activeagent at steady state (C_(av)ss).

In an embodiment of the invention directed to a patient in need ofremyelination, target peak serum levels of GGF2 are about 0.01 nM.

In an embodiment of the invention directed to a patient in need ofremyelination, target peak serum levels of GGF2 are at or about any ofthe following values, or range between the following values from about0.001 to 0.01 ng/ml; 0.01 to 0.1 ng/ml; 0.1 to 1.0 ng/ml; 1.0 to 10ng/ml; 10 to 100 ng/ml; or 100 to 1000 ng/ml. In a particularembodiment, the target peak serum level is about 1.0 ng/ml.

In an embodiment of the invention directed to a patient who has had astroke, target peak serum levels of GGF2 are at or about any of thefollowing values, or range between the following values from about0.00001 to 0.0001 ng/ml; 0.0001 to 0.001 ng/ml; 0.001 to 0.01 ng/ml;0.001 to 0.01 ng/ml; 0.01 to 0.1 ng/ml; 0.1 to 1.0 ng/ml; 1.0 to 10ng/ml; 10 to 100 ng/ml; 100 to 1000 ng/ml; 1000 to 10000 ng/ml; or 10000to 100000 ng/ml. In a particular embodiment, the target peak serum levelis about 0.2 micrograms/ml.

In an embodiment of the invention directed to a patient who hasneuropathy, target peak serum levels of GGF2 are at or about any of thefollowing values, or range between the following values from about 0.001to 0.01 ng/ml; 0.01 to 0.1 ng/ml; 0.1 to 1.0 ng/ml; 1.0 to 10 ng/ml; 10to 100 ng/ml; or 100 to 1000 ng/ml. In a particular embodiment, thetarget peak serum level is about 6.25 ng/ml.

In an embodiment of the invention directed to a patient who has heartfailure, target peak serum levels of GGF2 are at or about any of thefollowing values, or range between the following values from about 0.001to 0.01 ng/ml; 0.01 to 0.1 ng/ml; 0.1 to 1.0 ng/ml; 1.0 to 10 ng/ml; 10to 100 ng/ml; or 100 to 1000 ng/ml. In a particular embodiment, thetarget peak serum level is about 6.8 micrograms/ml.

In accordance with the present invention, pharmaceutical compositionscomprising GGF2 may be administered via different routes known to thoseskilled in the art. Any appropriate route of administration may beemployed, for example, intravenous, parenteral, subcutaneous,intramuscular, intracranial, intraorbital, ophthalmic, intraventricular,intracapsular, intraspinal, intracisternal, intraperitoneal, intranasal,aerosol, oral, or topical (e.g., by applying an adhesive patch carryinga formulation capable of crossing the dermis and entering thebloodstream) administration. Oral administration is envisioned toinclude sustained release oral dosage forms comprising GGF2. A GGF2pharmaceutical composition, as described herein, can be used to treatindividuals affected with neurological disorders wherein saidpharmaceutical composition maximizes the therapeutic effect, whileminimizing adverse side effects.

In a first embodiment of the present invention, GGF2 is administered toa mammal afflicted with a neurological disorder associated withdemyelination, wherein the GGF2 is administered in a dosing regimen toachieve and maintain a narrow target therapeutic window of plasma GGF2concentrations. As taught herein, precise dosing of GGF2 is necessary inorder to achieve serum plasma levels of GGF2 required for therapeuticefficacy with respect to inducing myelination in a subject in needthereof. Examples of demyelinating disorders for which suitable dosingof GGF2 is necessary in order to achieve therapeutic efficacy includeGuillain-Barre Syndrome, chronic inflammatory demyelinatingpolyneuropathy, peripheral demyelination due to traumatic injury,multiple sclerosis, optic neuritis, central demyelination due totraumatic injury, transverse myelitis, progressive multifocalleukoencephalopathy, Devic's disease (neuromyelitis optica), acutedisseminated encephalomyelitis, adrenoleukodystrophy andadrenoleukoneuropathy.

In a second embodiment of the present invention, GGF2 is administered toa mammal afflicted with a cardiac muscle disorder, such as congestiveheart failure, myocardial infarction, reperfusion injury, chemical,viral or idiopathic cardiotoxicity, arrhythmias, wherein the GGF2 isadministered in a dosing regimen to achieve a target therapeutic windowof plasma GGF2 concentrations.

In a third embodiment of the present invention, GGF2 is administered toa mammal that has suffered a stroke, spinal cord injury or traumaticbrain injury, wherein the GGF2 is administered in a dosing regimen toachieve a target therapeutic window of plasma GGF2 concentrations. Itwill be appreciated that for any of the applications detailed herein,GGF2 may be administered in any suitable form, or as a component in apharmaceutical composition and via any means, all of which are describedherein and/or understood in the art.

Accordingly, the present invention is directed to identification of atarget therapeutic window with respect to a therapeutically effectiveplasma level of GGF2. The target therapeutic window varies depending ofthe disease or disorder afflicting the patient and the desired activityconferred by achieving the appropriate therapeutically effective GGF2plasma level.

A method for selecting individuals based on presentation of symptoms isalso encompassed herein. Also encompassed is a method for selectingindividuals based on responsiveness to achieving the therapeuticallyeffective GGF2 plasma level, as indicated for each application, is alsoencompassed herein.

In addition to the methods of treatment set forth above, the presentinvention extends to the use of any of the compounds of the inventionfor the preparation of medicaments or as medicaments that may beadministered for such treatments, as well as to such compounds for thetreatments disclosed and specified.

The present invention also encompasses a pharmaceutical compositioncomprising GGF2 or an EGFL domain and a Mek1/Erk pathway inhibitor andits use in the treatment of a patient afflicted with a disease ordisorder associated with reduced levels of myelination.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C. FIG. 1A shows that GGF2-induced Akt and MAPK activation inSchwann cell-DRG neuron co-cultures. Schwann cell-DRG co-cultures undermyelinating condition were treated GGF (0.6 μM) and 20 minutes later,Akt and MAPK activation levels were assessed by Western blot analysis.FIG. 1B shows that inhibition of GGF2-induced MAPK activation by U0125.Co-cultures were pretreated with increasing doses of U0125 for 30minutes then stimulated with GGF2. Control cultures were left untreated.MAPK activation was assessed 20 minutes later. FIG. 1C shows thatinhibition of GGF2-induced MAPK activation by U0125 (1 and 3 μM)reverses the inhibitory effect of GGF2 on myelination. Co-cultures wereco-treated with GGF2 and U0125 (1 and 3 μM) under myelinatingconditions. Ten to twelve days later, cultures were fixed andimmunostained for MBP to assess the level of myelination.

FIGS. 2A-2B show that GGF2 promotes myelination at low concentrations.Co-cultures were treated with GGF2 at concentrations ranging from 0.5 to1000 pM (0.0005 to 1 nM) under myelinating conditions. Ten to twelvedays later, myelination was assessed by MBP immunostaining. Moreparticularly, the GGF2 concentrations from left to right are as follows:NT, 0.5 pM, 1 pM, 3 pM, 10 pM, 30 pM, 300 pM, 600 pM, and 1,000 pM,respectively (FIG. 2B). Ten to twelve days later, myelination wasassessed by MBP immunostaining (FIG. 2A).

FIGS. 3A-3F show that an inhibitory effect of GGF on myelination ismediated by the Mek1/Erk activation. FIG. 3A shows that Schwann cell DRGco-cultures treated with GGF (0.01, 0.6, and 1 nM) and 45 minutes laterthe cell lysates were prepared and levels of active Erk (p-Erk) and Akt(p-Akt) were determined by Western blot analysis. At I nM (boxed), GGFinduced activation of both Erk and Akt. FIG. 3B shows that inhibition ofGGF-induced Erk activation in co-cultures. Schwann cell-DRG co-cultureswere pre-treated with U0126 for 30 minutes then GGF (0.6 nM) was addedin the continuous presence of U0126. After 45 minutes the cell lysateswere prepared and level of p-Erk and p-Akt were determined. Treatmentwith U0126 inhibited both endogenous and GGF-induced Erk activationwithout affecting Akt activation. FIG. 3C shows images of MBP+myelinsegments formed in co-cultures treated with GGF or GGF+U0126 (1 nM).Treatment with U0126 abolished the inhibitory effect of GGF and inducedmyelination. Control cultures were maintained without any treatment(NT). Scale bar: 100 μm. Quantification of the result is shown in FIG.3D. FIG. 3E shows that inhibition of endogenous Erk activity inco-cultures promotes myelination. Co-cultures were treated withincreasing concentration of U0126 (0.5, 1 and 3 nM) under myelinatingcondition and 11 days later, myelination was analyzed as above. Asignificant increase in myelination was observed in cultures treatedwith U0126. Error bars indicate±SE (p<0.001). FIG. 3F shows thatinhibition of GGF-induced Erk activation is accompanied by a decrease inc-Jun and an increase in Krox 20 expression. Co-cultures were maintainedunder myelinating condition in the presence of GGF or GGF+U0126 (0.5, 1and 3 nM) for 11 days and the cell lysates were analyzed for MBP, c-Junand Krox 20 expression. Actin level served as a loading control.GGF-induced c-Jun expression was down-regulating with the treatment withU0126. Level of Krox 20 protein appeared increased in cultures treatedwith U0126.

FIGS. 4A-4D show that GGF promotes myelination at low concentration.FIG. 4A shows Schwann cells treated with varying concentrations of GGFranging from 0.0003 to 10 nM and 20 minutes later the cell lysates wereprepared and the levels of Erk and Akt activation were analyzed byWestern blot (top) and densitometric analysis (bottom). An increase inAkt activation appeared at lower concentration range (boxed) compared toErk activation. FIG. 4B shows co-cultures treated with differentconcentrations of GGF (0.0005, 0.001, 0.003, 0.01, 0.03, 0.3, 0.6, and 1nM) for 11 days under myelinating condition then fixed and immunostainedfor MBP and DAPI. Images of the control and cultures treated with 0.01nM of GGF are shown along with the quantification of the result (right).A clear biphasic effect of GGF is shown that promotes myelination at lowconcentrations (0.0005 to 0.01 nM) while inhibiting the process athigher (0.3 nM and above) concentration. FIG. 4C shows that lowconcentration of GGF (0.01 nM) significantly increased myelination onCRD-Nrg1^(+/−) neurons (p=0.003). Error bars show±SEM. Data wereanalyzed by one-way ANOVA (*: p<0.001). FIG. 4D is a bar graph showingpromyelinating effect of GGF2 in CRD-Nrg1^(+/−) co-cultures in which lowdoses of GGF2 rescued the myelination defect on the mutant axons.

FIGS. 5A-5D show the nucleic and amino acid sequences, designated SEQ IDNOs: 1 and 2, respectively, of full length GGF2.

FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10, and FIG. 11 show the nucleicand amino acid sequences of epidermal growth factor like (EGFL) domains1-6. The nucleic and amino acid sequences of the EGFL domains aredesignated as follows: SEQ ID NOs: 3 and 4 for EGFL domain 1 (FIG. 6);SEQ ID NOs: 5 and 6 for EGFL domain 2 (FIG. 7); SEQ ID NOs: 7 and 8 forEGFL domain 3 (FIG. 8); SEQ ID NOs: 9 and 10 for EGFL domain 4 (FIG. 9);and SEQ ID NOs: 11 and 12 for EGFL domain 5 (FIG. 10); and SEQ ID NOs:13 and 14 for EGFL domain 6 (FIG. 11).

FIG. 12 shows a table relating to neuregulin nomenclature.

DETAILED DESCRIPTION OF THE INVENTION

The data presented herein demonstrated that in order to promotemyelination of peripheral nerves, GGF2 must be administered to a mammalusing a dosing regimen directed to achieving a therapeutic window of,e.g., plasma GGF2 concentrations or GGF2 doses.

Definitions

The terms used herein have meanings recognized and known to those ofskill in the art, however, for convenience and completeness, particularterms and their meanings are set forth below. As used herein “about”means a stated value plus or minus another amount; thereby establishinga range of values. In certain preferred embodiments “about” indicates arange relative to a base (or core or reference) value or amount plus orminus up to 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.75%, 0.5%, 0.25% or 0.1%.

By “epidermal growth factor-like domain” or “EGF-like domain” is meant apolypeptide motif encoded by the NRG-1, NRG-2, or NRG-3 gene that bindsto and activates ErbB2, ErbB3, ErbB4, or combinations thereof, and bearsa structural similarity to the EGF receptor-binding domain as disclosedin Holmes et al., Science 256:1205-1210, 1992; U.S. Pat. No. 5,530,109;U.S. Pat. No. 5,716,930; U.S. Ser. No. 08/461,097; Hijazi et al., Int.J. Oncol. 13:1061-1067, 1998; Chang et al., Nature 387:509-512, 1997;Carraway et al., Nature 387:512-516, 1997; Higashiyama et al., JBiochem. 122:675-680, 1997; and WO 97/09425). See FIG. 6, FIG. 7, FIG.8, FIG. 9, FIG. 10, and FIG. 11 for nucleic and amino acid sequences ofepidermal growth factor like (EGFL) domains 1-6.

By “neuregulin” or “NRG” is meant a polypeptide that is encoded by anNRG-1, NRG-2, or NRG-3 gene or nucleic acid (e.g., a cDNA), and binds toand activates ErbB2, ErbB3, or ErbB4 receptors, or combinations thereof.

By “neuregulin-1,” “NRG-1,” “heregulin,” “GGF2,” or “p185erbB2 ligand”is meant a polypeptide that binds directly to or transactivates theErbB2 receptor and is encoded by the p185erbB2 ligand gene described inU.S. Pat. No. 5,530,109; U.S. Pat. No. 5,716,930; and U.S. Pat. No.7,037,888, the contents of each of which are incorporated herein byreference. See FIG. 6, 7, FIG. 8, FIG. 9, FIG. 10, and FIG. 11 for thenucleic and amino acid sequences of full length GGF2. See FIG. 12 for atable pertaining to neuregulin nomenclature.

Polypeptides encoded by the NRG-1, NRG-2, and NRG-3 genes possessEGF-like domains that allow them to bind to and activate ErbB receptors.Holmes et al. (Science 256:1205-1210, 1992) have shown that the EGF-likedomain alone is sufficient to bind and activate the p185erbB2 receptor.Accordingly, any polypeptide product encoded by the NRG-1, NRG-2, orNRG-3 gene, e.g., a polypeptide having an EGF-like domain encoded by aneuregulin gene or cDNA (e.g., an EGF-like domain, as described in U.S.Pat. No. 5,530,109; U.S. Pat. No. 5,716,930; U.S. Pat. No. 7,037,888,U.S. Pat. No. 7,135,456, and U.S. Pat. No. 7,319,019; or an EGF-likedomain as disclosed in WO 97/09425) may be used in the methods of theinvention to achieve a therapeutic window wherein an efficacious serumplasma level of GGF2 is achieved.

It must also be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of the present invention, particular methods, devices, andmaterials are now described.

“Local administration” means direct administration by a non-systemicroute at or near the site of affliction or disorder.

The terms “patient” and “subject” are used herein to refer to allanimals, including mammals. Examples of patients or subjects includehumans, cows, dogs, cats, goats, sheep, and pigs.

The term “pharmaceutically acceptable salts, esters, amides, andprodrugs” as used herein refers to those carboxylate salts, amino acidaddition salts, esters, amides, and prodrugs of the compounds of thepresent invention which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of patients without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe invention.

The term “prodrug” refers to compounds that are rapidly transformed invivo to yield the parent compounds of the above formula, for example, byhydrolysis in blood. A thorough discussion is provided in T. Higuchi andV. Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987, both of which are incorporated herein by reference.

The term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of compounds of the present invention. Thesesalts can be prepared in situ during the final isolation andpurification of the compounds or by separately reacting the purifiedcompound in its free base form with a suitable organic or inorganic acidand isolating the salt thus formed. Representative salts include thehydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate,oxalate, valerate, oleate, palmitate, stearate, laurate, borate,benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate,succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionateand laurylsulphonate salts, and the like. These may include cationsbased on the alkali and alkaline earth metals, such as sodium, lithium,potassium, calcium, magnesium, and the like, as well as non-toxicammonium, tetramethylammonium, tetramethylammonium, methylamine,dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.(See, for example, S. M. Barge et al., “Pharmaceutical Salts,” J. Pharm.Sci., 1977, 66:1-19 which is incorporated herein by reference.).

A “therapeutically effective amount” is an amount sufficient to decreasethe symptoms associated with a medical condition or infirmity, tonormalize body functions in disease or disorders that result inimpairment of specific bodily functions, or to provide improvement inone or more of the clinically measured parameters of a disease.Preferably, improvement in symptoms associated with the diseaseassociated with a demyelinating disease, for example, including walkingspeed, lower extremity muscle tone, lower extremity muscle strength, orspasticity. As related to the present application, a therapeuticallyeffective amount is an amount sufficient to reduce the pain orspasticity associated with the neurological disorder being treated, oran amount sufficient to result in improvement of sexual, bladder orbowel function in subjects having a neurological disorder which impairsnerve conduction; or which hinders normal sexual, bladder or bowelfunctions.

“Treatment” refers to the administration of medicine or the performanceof medical procedures with respect to a patient, to ameliorate theclinical condition of the patient, including a decreased duration ofillness or severity of illness, or subjective improvement in the qualityof life of the patient or a prolonged survival of the patient.

As used herein, the term “target therapeutic window” refers to the doserange or serum concentration range that achieves the desired therapeuticresults. With regard to GGF2, in a particular embodiment, the targettherapeutic window refers to an amount of GGF2 sufficient to induceSchwann cell myelination in a subject, which amount is less than theamount sufficient to inhibit myelination in a subject. In a surprisingdiscovery, the present inventors identified the target therapeuticwindow for GGF2 with respect to its ability to promote myelination bydetermining the relative levels of P13-kinase pathway activation andMek1/Erk pathway activation. More particularly, the present inventorsdiscovered the hitherto unrealized positive correlation betweenGGF2-mediated PI3-kinase pathway activation and promotion of myelinationand a negative correlation between GGF2-mediated Mek1/Erk pathwayactivation and promotion of myelination. Alternatively stated, thepresent inventors discovered that administration of GGF2 can be finelytuned to promote myelination by assessing activation levels of thesepathways. A target therapeutic window for GGF2 with regard to promotingmyelination in a subject is defined as an amount of GGF2 that promotesPI3-kinase pathway activation (assayed, for example, by detectingphosphorylated Akt) in the absence of detectable Mek1/Erk pathwayactivation (assayed, for example, by detecting phosphorylated Erk).Detection of phosphorylated Akt and phosphorylated Erk can be achievedusing standard assays known in the art, including ELISA, Western(immuno) blot, immunocyto chemistry, in vitro kinase assay, LC/MS(liquid chromatography/mass spectrometry), MaldiTOF MS (Matrix AssistedLaser Desorption/Ionization—Time of Flight mass spectrometry) or otherprotein systems known to the field such as Luminex

One skilled in the art would appreciate that other intracellular markersof PI3-kinase pathway activation and Mek1/Erk pathway activation areknown and are used in accordance with the present invention. Inaccordance with the present invention, other indicators of P13-kinasepathway activation and Mek1/Erk pathway activation can be used todetermine the therapeutic window in which GGF2 promotes myelination.

In addition, the compounds of the present invention can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms for the purposesof the present invention.

“MAP Kinase Inhibitors”

A non-limiting list of MAP kinase inhibitors that may be used in thepresent invention includes: Arctigenin, which potently inhibits theactivity of MKK1 in vitro with an 1050 value of 1 nM and thus inhibitsthe phosphorylation and activation of MAP kinases ERK1/2, p38 kinase andJNK and their activities in Raw264.7 cells treated with LPS; PD 98059,which is a potent, selective and cell-permeable inhibitor of MAPkinase-kinase (also known as MAPKJERK kinase or MEK) that inhibitsphosphorylation of MAP kinase by MAP kinase-kinase but does not inhibitMAP kinase itself. The IC50 values for PD 98059-induced effects are inthe 1-20 μM range for many assays; SB202190, which is a highlyselective, potent and cell permeable inhibitor of p38 MAP kinases thatbinds within the ATP pocket of the active kinase with a Kd of 38 nM asmeasured in recombinant human p38 and selectively inhibits the p38alphaand beta isoforms (IC50 values are 50 and 100 nM for p38alpha/SAPK2alphaand p38beta2/SAPK2beta respectively); SB203580,which is a highlyselective and cell permeable inhibitor of p38 mitogen-activated proteinkinase with IC50 values of 50 and 500 nM for p38/SAPK2a and p38/SAPK2brespectively and also inhibits the phosphoinositide-dependent proteinkinase 1 (PDK1) at 10-fold higher concentrations (IC50 ˜3-5 μM)(Displays 100-500-fold selectivity over Lck, GSK3b and Akt/PKB); SL 327,which is a selective inhibitor of MEK1 and MEK2 with IC50 values of 180and 220 nM, respectively. It blocks hippocampal LTP in vitro and isbrain penetrant in vivo, blocking fear conditioning and learning inrats, and producing neuroprotection in mice, following systemicadministration; SP600125, which is a selective inhibitor of c-JunN-terminal kinase (JNK). It competitively and reversibly inhibits JNKI,2 and 3 (IC50=40-90 nM) and has been shown to have less inhibitorypotency on ERK2, p38b and a range of other kinases and is known to beactive in vivo; and U0126, which is a selective inhibitor of themitogen-activated protein kinase kinases, MEK-1 and MEK-2, with a100-fold higher potency than PD 98059 and is a weak inhibitor of PKC,Raf, ERK, JNK, MEKK, MKK-3, MKK-4/SEK, MKK-6, Abl, Cdk2 and Cdk4 andinhibits AP-1 transactivation in cell-based reporter assays.

Other inhibitors that are currently in FDA Phase trial include thefarnesyl transferase inhibitors (FTIs). Zarnestra® (R115777,tipifarnib), for example, is the FTI that is furthest along indevelopment. A phase II trial of patients with previously treatedmetastatic breast cancer tested two different dosing schedules:continuous and intermittent. The objective response rates in the 2groups were 10% and 14%, with an additional 15% and 9% who had stabledisease for at least 6 months. The major side effects observed were bonemarrow suppression and neuropathy, both of which were less in theintermittent dosing group than the continuous. Several phase I studiesof zarnestra and other FTIs have been performed in combination withcytotoxic chemotherapy and have demonstrated the safety of thesecombination regimens. Phase II trials in breast cancer are underway,including one using zamestra in combination with an aromatase inhibitor.FDA approval for zarnestra use in acute myeloid leukemia (AML) ispending phase III data, as the FDA committee voted against acceleratedapproval for zarnestra based on data from a single-armed phase II trial.

With regard to Zarnestra®, for Phase I clinical trials, Zarnestra® isadministered at 400 mg administered orally twice daily for two weeks;for Phase II clinical trials, Zarnestra® is administered at 300 mgadministered orally twice daily for the first 21 days of each 28-daycycle; for Phase III clinical trials, Zarnestra® is administered at 600mg administered orally twice daily for the first 21 days of each 28-daycycle.

The Raf inhibitors comprise another types of inhibitors that arecurrently in FDA Phase trials. Sorafenib (BAY 43-9006), for example, isthe first compound to target not only the Raf/MEKJErk signaling pathway,but also the VEGFR and PDGFR pathways. In March 2004, sorafenib wasgranted Fast Track status by the FDA for metastatic renal cell cancer.In April 2005, sorafenib was accepted into the Pilot 1 Program, which isdesigned for therapies that have been granted FDA Fast Track status andthat have the potential to provide significant benefit over existingstandard therapy. There are also several large, international,multi-institution phase III clinical studies of sorafenib underway inpatients with advanced stage primary cancers of the kidney and liver, aswell as metastatic melanoma.

With regard to Sorafenib, Phase I clinical trials tested two doselevels: Dose Level 1: 200 mg of Sorafenib by mouth twice a day for a 3week cycle or Dose Level 2: 400 mg of Sorafenib by mouth twice a day fora 3 week cycle.

The results of a planned interim analysis of an ongoing phase III trialin patients with advanced kidney cancer were recently presented(Escudier et al. J Natl Cancer Inst. 2008 100:1454-63; the contents ofwhich are incorporated herein in their entirety). Among 769 analyzedpatients, progression-free survival (PFS) was doubled to a median valueof 24 weeks with sorafenib, compared to 12 weeks with placebo. Thebenefits from sorafenib were observed in all patient subgroups,regardless of age, duration of disease, or prior therapies. Diseasecontrol was achieved in 80% of patients who received sorafenib: 78% hadstable disease (compared to 55% in the placebo arm) and 2% had partialresponse (compared to none in the placebo arm). The 12-weekprogression-free rate was 79% for sorafenib vs. 50% for placebo.Furthermore, sorafenib was very well tolerated in 768 patients, and themost common side effects were hypertension, fatigue, diarrhea, and rash,including a rash on the hand and foot (hand and foot syndrome). Phase Hefficacy trials are studying sorafenib as a single agent in advancedlung, breast, and other cancers. Phase I/II clinical trials areinvestigating sorafenib in combination with a range of standardchemotherapeutics and other anticancer agents.

ISIS 5132 is another raf inhibitor that has shown acceptable toxicity inphase I studies. Phase II studies are now underway in a variety ofcancer types.

Other inhibitors that arc currently in FDA Phase trial include the MEKinhibitors. CI-1040, for example, is an oral, selective small-moleculeinhibitor of MEK 1-2. Animal and culture studies have shown activity ofthis agent in breast cancer cell lines. Phase I studies have found mildgastrointestinal and skin side effects. Unfortunately, a phase H studyin 67 patients with 4 different tumor types (advanced colorectal, NSCLC,breast, and pancreatic cancer) found no responses, although CI-1040treatment was well tolerated.

PD 0325901, a second generation MEK inhibitor, has recently enteredclinical development and appears to have noticeably better pharmacologicproperties compared to CI-1040, which investigators hope may translateinto better anti-cancer efficacy. It has shown some partial response inmelanoma patients.

With regard to PD 0325901, Phase I and Phase II clinical trials testedmultiple dose levels. Administered orally either once or twice a day;several dosing schedules evaluated; current dosing schedule 5 dayson-drug, 2-days off drug for 3 weeks in a 28-day cycle. Doses evaluatedranged from 1 mg once a day to 30 mg twice daily. Clinical trials wereprematurely discontinued due to safety concerns, specifically ocular andneurological toxicity presented at 10 mg twice-a-day and higher doses.

It is to be understood that this invention is not limited to theparticular molecules, compositions, methodologies or protocolsdescribed, as these may vary. It is also to be understood that theterminology used in the description is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is limited only by the appended claims.

Administration: Neuregulins and polypeptides containing EGF-like domainsencoded by neuregulin genes may be administered to patients orexperimental animals with a pharmaceutically-acceptable diluent,carrier, or excipient, in unit dosage form. Conventional pharmaceuticalpractice may be employed to provide suitable formulations orcompositions to administer such compositions to patients or experimentalanimals. Any appropriate route of administration may be employed, forexample, intravenous, parenteral, subcutaneous, intramuscular,intracranial, intraorbital, ophthalmic, intraventricular, intracapsular,intraspinal, intracisternal, intraperitoneal, intranasal, aerosol, oral,or topical (e.g., by applying an adhesive patch carrying a formulationcapable of crossing the dermis and entering the bloodstream)administration. Therapeutic formulations may be in the form of liquidsolutions or suspensions; for oral administration, formulations may bein the form of tablets or capsules; and for intranasal formulations, inthe form of powders, nasal drops, or aerosols. Any of the aboveformulations may be in a sustained-release formulation.

Methods well known in the art for making formulations are found in, forexample, “Remington's Pharmaceutical Sciences.”, which is incorporatedherein in its entirety. Formulations for parenteral administration may,for example, contain excipients, sterile water, or saline, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin, orhydrogenated napthalenes. Sustained-release, biocompatible,biodegradable lactide polymer, lactide/glycolide copolymer, orpolyoxyethylene-polyoxypropylene copolymers may be used to control therelease of the compounds. Other useful parenteral delivery systems foradministering molecules of the invention include ethylene-vinyl acetatecopolymer particles, osmotic pumps, implantable infusion systems, andliposomes. Formulations for inhalation may contain excipients, forexample, lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may beoily solutions for administration in the form of nasal drops, or as agel.

Thus, and as stated earlier, the present invention includes within itsscope, and extends to, the recited methods of treatment and to the useof such compounds for the preparation of medicaments useful for suchmethods.

Demyelinating Diseases: Myelin sheaths cover many nerve fibers in thecentral and peripheral nervous system. The presence of intact myelinsheaths accelerates axonal transmission of neural impulses. Disordersthat affect myelin interrupt nerve transmission and disease symptoms mayreflect deficits in any part of the nervous system.

Myelin formed by oligodendroglia in the central nervous system (CNS)differs chemically and immunologically from that formed by Schwann cellsperipherally. Thus, some myelin disorders (e.g., Guillain-Barresyndrome, chronic inflammatory demyelinating polyneuropathy, and otherperipheral nerve polyneuropathies) tend to affect primarily theperipheral nerves, whereas other myelin disorders affect primarily theCNS. The most commonly affected areas in the CNS are the brain, spinalcord, and optic nerves.

Demyelination is often secondary to an infectious, ischemic, metabolic,or hereditary disorder. In primary demyelinating disorders, while thecause or causes are unknown, an autoimmune mechanism is suspectedbecause the disorder sometimes follows a viral infection or viralvaccination.

Demyelination tends to be segmental or patchy, affecting multiple areassimultaneously or sequentially. Remyelination can occur, however, withrepair, regeneration, and complete recovery of neural function.Extensive myelin loss, however, is usually followed by axonaldegeneration and often cell body degeneration.

Multiple sclerosis (MS) is characterized by disseminated patches ofdemyelination in the brain and spinal cord. Common symptoms includevisual and oculomotor abnormalities, paresthesias, weakness, spasticity,urinary dysfunction, and mild cognitive impairment. Typically,neurologic deficits are multiple, with remissions and exacerbationsgradually producing disability. Diagnosis is by history of remissionsand exacerbations plus clinical signs, test results, lesions seen onmagnetic resonance imaging (MRI), or other criteria (depending onsymptoms) to objectively demonstrate >2 separate neurologicabnormalities. Treatment generally includes corticosteroids for acuteexacerbations, immunomodulatory drugs to prevent exacerbations,andsupportive measures.

In MS, localized areas of demyelination (plaques) occur, withdestruction of oligodendroglia, perivascular inflammation, and chemicalchanges in lipid and protein constituents of myelin in and around theplaques. Axonal damage is possible, but cell bodies and axons tend to berelatively well preserved. Fibrous gliosis develops in plaques that aredisseminated throughout the CNS, primarily in white matter, particularlyin the lateral and posterior columns (especially in the cervicalregions), optic nerves, and periventricular areas. Tracts in themidbrain, pons, and cerebellum are also affected. Gray matter in thecerebrum and spinal cord can be affected, but to a much lesser degree.

Heart Disease

Heart disease is a general term for a number of different diseases whichaffect the heart. It is the leading cause of death in manyindustrialized countries, including the United States. The followingbroad categories of heart disease are presented by way of introduction.Extrinsic cardiomyopathies are cardiomyopathies, wherein the primarypathology lies outside the myocardium. Most cardiomyopathies areextrinsic, because the most common cause of cardiomyopathy is ischemia.Intrinsic cardiomyopathies derive from weakness in the heart muscle thatis not due to an identifiable external cause. Cardiovascular disease, onthe other hand, refers to any number of specific diseases that affectthe heart itself and/or the blood vessel system, especially the veinsand arteries leading to and from the heart. Research on diseasedimorphism suggests that women who suffer with cardiovascular diseaseusually suffer from forms that affect the blood vessels while menusually suffer from forms that affect the heart muscle itself. Known orassociated causes of cardiovascular disease include diabetes mellitus,hypertension, hyperhomocysteinemia and hypercholesterolemia. Ischaemicheart disease is yet another category of disease of the heart itself,typified by reduced blood supply to the organ.

Hypertensive heart disease is a term used to refer to heart diseasecaused by high blood pressure, especially localized high blood pressureInflammatory heart disease involves inflammation of the heart muscleand/or the tissue surrounding it. Valvular heart disease is any diseaseprocess involving one or more valves of the heart. The valves in theright side of the heart are the tricuspid valve and the pulmonic valveand the valves in the left side of the heart are the mitral valve andthe aortic valve.

Congestive heart failure, one of the leading causes of death inindustrialized nations, results from an increased workload on the heartand a progressive decrease in its pumping ability. It can result fromany structural or functional cardiac disorder that impairs the abilityof the heart to fill with or pump a sufficient amount of blood throughthe body. Initially, the increased workload that results from high bloodpressure or loss of contractile tissue induces compensatorycardiomyocyte hypertrophy and thickening of the left ventricular wall,thereby enhancing contractility and maintaining cardiac function. Overtime, however, the left ventricular chamber dilates, systolic pumpfunction deteriorates, cardiomyocytes undergo apoptotic cell death, andmyocardial function progressively deteriorates.

Factors that underlie congestive heart failure include high bloodpressure, ischemic heart disease, exposure to cardiotoxic compounds suchas the anthracycline antibiotics, and genetic defects known to increasethe risk of heart failure.

By “congestive heart failure” is meant impaired cardiac function thatrenders the heart unable to maintain the normal blood output at rest orwith exercise, or to maintain a normal cardiac output in the setting ofnormal cardiac filling pressure. A left ventricular ejection fraction ofabout 40% or less is indicative of congestive heart failure (by way ofcomparison, an ejection fraction of about 60% percent is normal).Patients in congestive heart failure display well-known clinicalsymptoms and signs, such as tachypnea, pleural effusions, fatigue atrest or with exercise, contractile dysfunction, and edema. Congestiveheart failure is readily diagnosed by well known methods (see, e.g.,“Consensus recommendations for the management of chronic heart failure.”Am. J. Cardiol., 83(2A): IA-38-A, 1999).

Relative severity and disease progression are assessed using well knownmethods, such as physical examination, echocardiography, radionuclideimaging, invasive hemodynamic monitoring, magnetic resonanceangiography, and exercise treadmill testing coupled with oxygen uptakestudies.

By “ischemic heart disease” is meant any disorder resulting from animbalance between the myocardial need for oxygen and the adequacy of theoxygen supply. Most cases of ischemic heart disease result fromnarrowing of the coronary arteries, as occurs in atherosclerosis orother vascular disorders.

By “myocardial infarction” is meant a process by which ischemic diseaseresults in a region of the myocardium being replaced by scar tissue.

By “cardiotoxic” is meant a compound that decreases heart function bydirecting or indirectly impairing or killing cardiomyocytes.

By “hypertension” is meant blood pressure that is considered by amedical professional (e.g., a physician or a nurse) to be higher thannormal and to carry an increased risk for developing congestive heartfailure.

By “treating” is meant that administration of a neuregulin orneuregulin-like polypeptide slows or inhibits the progression ofcongestive heart failure during the treatment, relative to the diseaseprogression that would occur in the absence of treatment, in astatistically significant manner. Well known indicia such as leftventricular ejection fraction, exercise performance, and other clinicaltests, as well as survival rates and hospitalization rates may be usedto assess disease progression. Whether or not a treatment slows orinhibits disease progression in a statistically significant manner maybe determined by methods that are well known in the art (see, e.g.,SOLVD Investigators, N. Engl. J. Med. 327:685-691, 1992 and Cohn et al.,N. Engl. J Med. 339:1810-1816, 1998).

By “decreasing progression of myocardial thinning” is meant maintaininghypertrophy of ventricular cardiomyocytes such that the thickness of theventricular wall is maintained or increased.

By “inhibits myocardial apoptosis” is meant that neuregulin treatmentinhibits death of cardiomyocytes by at least 10%, more preferably by atleast 15%, still more preferably by at least 25%, even more preferablyby at least 50%, yet more preferably by at least 75%, and mostpreferably by at least 90%, compared to untreated cardiomyocytes.

Stroke

Stroke or cerebrovascular accident (CVA) is a term used to refer to therapidly developing loss of brain functions due to a disturbance in theblood vessels supplying blood to the brain. A stroke occurs when theblood supply to part of the brain is suddenly interrupted or when ablood vessel in the brain bursts, spilling blood into the spacessurrounding brain cells. Brain cells die when they no longer receiveoxygen and nutrients from the blood or there is sudden bleeding into oraround the brain. The symptoms of a stroke include sudden numbness orweakness, especially on one side of the body; sudden confusion ortrouble speaking or understanding speech; sudden trouble seeing in oneor both eyes; sudden trouble with walking, dizziness, or loss of balanceor coordination; or sudden severe headache with no known cause. Thereare two forms of stroke: ischemic, which is due to blockage of a bloodvessel supplying the brain (e.g., caused by thrombosis or embolism); andhemorrhagic, which results from bleeding into or around the brain.

Index for Therapeutic Window

For each disease application described herein, a target therapeuticwindow for GGF2 serum plasma levels is established. In accordance withthe experimental results presented herein, when GGF2 is administered toa mammal afflicted with a neurological disorder associated withdemyelination, GGF2 must be administered in a dosing regimen to achieveand maintain a narrow target therapeutic window of plasma GGF2concentrations. As taught herein, precise dosing of GGF2 is necessary inorder to achieve serum plasma levels of GGF2 required for therapeuticefficacy with respect to inducing myelination in a subject in needthereof.

In an embodiment of the invention directed to a patient in need ofremyelination a particular embodiment, the target serum plasma level ofGGF2 is about 0.01 nM.

In another embodiment of the invention directed to a patient in need ofremyelination, GGF2 is administered at an amount of about 500 ng/kg ofpatient body weight.

The compositions of the present invention may be used in the treatmentof a condition in a patient that includes establishing a therapeuticallyeffective concentration of GGF2 in the patient in need thereof. Thecompositions may be used for building up a level and or maintaining atherapeutically effective concentration of GGF2 in the patient. Wheredesirable, the compositions of the present invention may be formulatedto avoid large peaks in initial release of GGF2. The compositions of thepresent invention when administered to a patient in need thereof providefor the treatment of the above-indicated diseases. Preferably, thecompositions are administered so as to achieve a therapeuticallyeffective blood plasma level of GGF2 that is maintained in the patientfor a period of at least 6 hours, preferably at least 8 hours, and morepreferably at least about 10-12.

EXAMPLES

Materials and Methods

Antibodies

For immunofluorescence analysis, monoclonal antibody (SM194) to myelinbasic protein (MBP) (Sternberger monoclonals) was used at a 1:500dilution. For Western blot analysis, polyclonal antibodies to activeerbB2 (p-Neu/Tyr 1248), erbB2 and erbB3 were all obtained from SantaCruz and used at a 1:1000 dilution. Monoclonal antibody tophosphorylated Akt and polyclonal antibody to phosphorylated MAPK werepurchased from Cell Signaling and were used at dilutions of 1:1000 and1:500, respectively. Polyclonal antibodies to Akt and MAPK (Promega)were used at dilutions of 1:1000 and 1:5000, respectively.

Type-II and Type-III Neuregulin-1

Recombinant human glial growth factor-II (rhGGF-II, Type-II Nrg1) wasobtained from Acorda Therapeutics, Inc. Recombinant human sensory andmotor neuron derived factor (rhSMDF, Type-III Nrg1) was purchased fromR&D Systems. In the present study, rhGGF-II and rhSMDF are referredsimply as GGF (or GGF2) and SMDF, respectively. The GGF was theN-terminus 419 amino acid residues containing the EGF domain and theIg-like domain. Accordingly, GOP is a soluble protein lacking atransmembrane and cytoplasmic domains.

Primary Rat Schwann Cell Culture

Schwalm cells were prepared from sciatic nerves of newborn rats (1-2 dayold) as described previously (Brookes et al., Brain Res.1979;165:105-118). For routine culture, Schwann cells were grown in.Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum(FBS) supplemented with EGF-domain neuregulin-1 (R&D Systems) (10 ng/ml)and Forskolin (2 μM). Cells between passage 2-4 were used in allexperiments described in the text.

Dorsal Root Ganglion (DRG) Neuron-Schwann Cell Co-culture

Dissociated DRG were prepared from embryonic day 14.5 rat embryos asdescribed previously (Eldridge et al., J Cell Biol. 1987;105(2):1023-34) and plated onto collagen (type-1 rat tailcollagen,)-coated 12 mm glass coverslips at a density of 1.25DRG/coverslip. Five to six hours later, the cultures were flooded withneurobasal medium (Cellgro,) supplemented with B27 (GIBCO), 20% glucose,NGF (50 ng/ml) and 5-fluorodeoxyuridine (FUdR, 10 μM) and maintained inthe medium for additional 2-3 days in order to remove proliferatingnon-neuronal cells. Cultures were then switched to fresh medium withoutFUdR and maintained until the DRG axons reached the periphery of thecoverslips. After the axonal networks were established, Schwann cellswere plated onto the neurons at a density of 100,000 cells/coverslip.Four to five days later, cultures were switched to myelinating medium:Minimal Essential Medium (MEM) supplemented with 10% heat-inactivatedFBS, 20% glucose, NGF (50 ng/ml) and ascorbic acid (50 μg/ml). Ten toeleven days later, myelination was assessed by immunostaining for MBP.

Superior Cervical Ganglion (SCG) Neuron-Schwann Cell Co-culture

Dissociated SCG were prepared from postnatal day 1-2 rats as describedpreviously and plated onto collagen-coated 12 mm glass coverslips at adensity of 0.8 SCG/coverslip. Next day, the cultures were flooded withneurobasal medium supplemented with B27 (GIBCO), 20% glucose, NGF (50ng/ml) and 5-fluorodeoxyuridine (FUdR, 10 μM) and maintained in themedium for an additional 2-3 days in order to remove proliferatingnon-neuronal cells. The cultures were switched back to fresh mediumwithout FudR and maintained until the axons extended out to theperiphery of the coverslips. Schwann cells were plated onto the neuronsand maintained in neurobasal medium with supplements until the Schwanncells populate the axons (about 7-10 days). Myelination was initiated byplacing the cultures in myelinating medium as described for DRG-Schwanncell co-culture. Forty days later, myelination was assessed by MBPimmunostaining.

Immunoprecipitation and Western Blot Analysis

To prepare cell lysates, 90-95% confluent rat Schwann cells on 60 mmplates or co-cultures were washed twice in phosphate-buffered saline(PBS) and then lysed in 300 μl ice-cold lysis buffer (50 mM Tris HCl pH7.4, 1% NP-40, 0.25% Sodium Deoxycholate, 150 mM NaCl, 1 mM EGTA, 10μg/ml Leupeptin, 2 μg/ml Aprotinin, 1 mM PMSF and 0.5 mM sodiumorthovanadate). Lysates were cleared by centrifugation for 15 min at14,000 rpm in the cold and the protein concentration of the supernatantswas determined according to manufacturer specifications (Bio-Rad:Hercules, Calif.). For Western blot analysis, 50-70 μg of Schwann celllysates were size-fractionated on 10% SDS-polyacrylamide gels andtransferred onto PVDF membranes. After blocking in 5% milk, themembranes were incubated with appropriate primary antibodies prepared inblocking solution. After incubating with horseradish peroxidaseconjugated secondary antibodies, the protein bands were visualized byenhanced chemiluminescence. For immunoprecipitation, 500 μg of Schwanncell lysates were incubated with 0.6 μg of primary antibody for 3 hoursat 4° C., then incubated with 50 μl Sepharose A beads for 1 hour. Beadswere washed 5 times in the lysis buffer and proteins bound to beads werefractionated on SDS-polyacrylamide gels and subjected to Western blotanalysis.

Immunofluorescence Staining for MBP

DRG-Schwann cell or SCG-Schwann cell cultures were rinsed in phosphatebuffered saline (PBS) then fixed in 4% paraformaldehyde for 20 minutes.After washing with PBS, samples were permeabilized in ice-cold methanolfor 25 minutes then incubated in blocking solution (5% normalgoat-serum+0.3% Triton X) for 1 hour at room temperature. This wasfollowed by incubation with primary antibody prepared in blockingsolution overnight. After washing with PBS, samples were incubated withAlexa-488 conjugated goat-anti-mouse secondary antibody for 45 minutes.Nuclei of cells were visualized by staining with DAPI.

Real-Time Quantitative PCR

Statistical Analysis

One way ANOVA was performed using SAS programming software with 95%significance level.

Results

The inhibitory function of GGF2 on myelination is mediated by the MAPKactivation An earlier study has shown that Nrg1 type II (GGF2), whenadded to Schwann cell-neuron co-cultures, inhibits myelination. It hasalso been reported that activation of Ras/Raf/MAPK pathway inhibitsmyelin-associated gene expression in Schwann cells, whereas activationof the PI3-kinase pathway promotes myelination, leading to a notion thatthe myelination state of Schwann cell is determined by the balancebetween PI3-kinase and Ras/Raf/MAPK pathways (Ogata et at J Neurosci2004;24:6724-32). The present inventors predicted that if GGF2 acts viaMAPK activation to inhibit myelination, inhibition of GGF2-induced MAPKactivation would reverse the inhibitory effect on myelination. To assessthe possibility that the inhibitory effect of GGF2 on myelination couldbe due to its ability to induce a robust MAPK activation in Schwanncells, the present inventors used a well-established in vitromyelinating culture system in which Schwann cells are co-cultured withdorsal root ganglion (DRG) neurons and induced to myelinate theassociated axons by addition of ascorbic acid to the culture media.First, to determine the effect of GGF2 on MAPK activation in theco-cultures, primary Schwann cells were plated onto DRG neurons andallowed to propagate the axons. Once the cultures stopped proliferating,the co-cultures were stimulated with GGF2 at 0.6 nM. Twenty minuteslater, cell lysates were prepared and MAPK activation was determined byWestern blot analysis. In control co-cultures, there was a low level ofactive MAPK. As shown in FIGS. 1A-1C, treatment with GGF2 furtherincreased the level of MAPK activation. To determine whether theGGF2-induced MAPK activation could be blocked by treatment with U0126, apharmacologic inhibitor of MAPK kinase, co-cultures were pretreated withincreasing concentrations (0.5, 1, 3 and 10 μM) of U0126 for 30 minutesprior to GGF2 stimulation and these concentrations were maintained inthe culture medium. Control cultures were treated with the inhibitor inthe absence of GGF2 treatment. In both control and experimentalcultures, U0126-mediated MAPK inhibition was concentration-dependent, asindicated by the progressive decrease in the levels of phospho-MAPK. Incultures treated with GGF2 and U0126 at a concentration of 1 μM, thelevel of activation was reduced to the basal level, while at 10 μMU0126, MAPK activation in the co-culture was completely abolished. TheU0126 had no effect on GGF2-induced P13kinase activation.

In order to evaluate the effect of MAPK inhibition on myelination,co-cultures were treated with GGF2 in the presence or the absence ofU0126 at the time of initiating myelination and the same conditions weremaintained under the described myelinating condition. Control cultureswere left untreated under the described myelinating condition. Ten toeleven days later, cultures were fixed and immunostained for myelinbasic protein (MBP) to visualize myelin segments. In cultures treatedwith GGF2, there was a marked decrease in the number of myelin segmentsas shown previously, revealing the inhibitory effect of GGF2 onmyelination. In cultures co-treated with U0126, however, there was adose-dependent increase in myelination, indicating that blocking MAPKactivation reversed the inhibitory effect of GGF2.

GGF2 promotes myelination at low concentrations: Although the level ofMAPK activation steadily increased in Schwann cells treated withincreasing concentrations of GGF2, the present inventors observed thatat low concentrations below 0.01 μM, while the level of Akt activationincreased significantly above the basal level, there was no detectablelevel of MAPK activation. If the myelination state of a Schwann cell isdetermined by the balance between the Akt and MAPK activation, thepresent inventors sought to evaluate if the increase in Akt activationin the absence of MAPK activity at these concentrations is correlatedwith a positive effect on myelination. To investigate this potentiality,co-cultures were treated with GGF2 at concentrations ranging from 0.0005and 0.03 nM at the time of initiating myelination. Cultures were laterfixed and immunostained for MBP. As predicted based on the instantfindings, there was an increase in the level of myelination in culturestreated with low doses of GGF2, ranging from 0.0005 to 0.01 nM, comparedto the untreated control cultures. When quantitated, the resultdemonstrated that there was a dose-dependent increase in the number ofmyelin segments (FIGS. 2A-2B): a 1.9-, 2.7-, and 3.5-fold increase inmyelination relative to the control level, at 0.0005, 0.001 and 0.01 nMGGF2, respectively. At 0.03 nM, there was a drastic decrease in thelevel of myelination to a level close to, or slightly below the controlcultures. Subsequent increases in the amount of GGF2 resulted in furtherdecreases in myelination. Myelination responsive to GGF2 was completelyinhibited at 0.6 nM GGF2. This concentration corresponded to theappearance of active MAPK in the co-cultures as shown in FIGS. 1A-C.These results suggest that GGF2 plays dual roles during myelination: onethat promotes and the other that inhibits myelination, and the twoopposing functions are determined by the dosage of GGF2 presented to theSchwann cells.

The opposing functions of GGF2 are mediated by Mek/Erk activation: Toinvestigate further the opposing functions of GGF2, additionalexperiments were performed. Previous studies have implicated Ras/Raf/Erkand PI-3 kinase, respectively, as negative and positive regulators ofmyelination, suggesting that a balance between the two is correlatedwith the myelination state of the Schwann cells. To delineate furtherthe activation states of the pathways induced by GGF2, co-cultures weretreated with the soluble GGF2 protein at 1 nM. The present inventorsdetermined that at this concentration, GGF2 effectively inhibitedmyelination. Cell lysates were prepared 30 minutes following the GGF2treatment and the presence of the phosphorylated proteins was determinedby Western blot analysis (FIG. 3A). At 1 nM (FIG. 3A, boxed lanes) GGF2increased Akt activation above the basal level. An increase in Erkactivation was also observed in GGF2-treated cultures at thisconcentration. Concentrations of GGF2 as low as 0.6 nM were shown to besufficient for Erk activation in GGF2 treated cultures.

To corroborate the above results and investigate further the correlativelink between Erk activation and the inhibitory effect of GGF onmyelination, additional experiments were performed. Accordingly,co-cultures were treated with GGF2 along with increasing concentrationsof U0126, the above-described specific inhibitor of Mek1/Erk pathway.Western blot analysis presented in FIG. 3B shows that U0126 inhibitedGGF2-induced Erk activation in a dosage-dependent manner while it had noeffect on Akt activation. The low level of endogenous Erk activitynormally observed in the co-culture system was also decreased with thedrug treatment.

The present inventors further assessed the effect of Mek1/Erk inhibitionon myelination. As shown in FIG. 3C and FIG. 3D, addition of GGF2 athigh concentration almost completely inhibited myelination in theco-cultures. In cultures co-treated U0126, however, the inhibitoryeffect of GGF2 was reversed, as indicated by the dosage-dependentincrease in the level of myelination (FIG. 3C and FIG. 3D). This resultprovides direct evidence that the inhibitory effect of GGF2 onmyelination is mediated by the Erk activation. Interestingly, U0126treatment in co-cultures in the absence of GGF2 also resulted in anincrease in the level of myelination (FIG. 3E), which indicates that theendogenous Mek1/Erk activity functions as an intrinsic negativeregulator of myelination.

Western blot analysis on lysates prepared from the co-cultures alsorevealed that GGF2 treatment increased expression of c-Jun protein, anegative regulator of Schwann cell differentiation and myelination.Subsequent inhibition of the GGF2-induced Mek1/Erk activitydown-regulated c-Jun levels, which, in turn, was accompanied by anincrease in myelin protein expression. Unlike the effect on c-Jun, U0126treatment resulted in an increase in Krox20 expression in theco-cultures. This is in agreement with a recent report suggesting across antagonistic relationship between c-Jun and Krox20 in regulatingmyelination (Parkinson et al, 2008, Journal of Cell Biology181:625-637).

GGF2 promotes Schwann cell myelination: To corroborate and extendresults presented herein and evaluate further the opposing functions ofGGF2, the present inventors assessed the concentration-dependent effectof GGF2 on Ras/Raf/Erk and PI3-kinase activation in Schwann cells. Cellswere treated with various concentrations of GGF2 ranging from 0.0003 to10 nM and the level of Erk and Akt activation was determined by Westernblot analysis. Images and the relative increase in the activation levelsare presented in FIG. 4A and FIG. 4B. The level of active Akt increasedsteadily beginning at the lowest dose tested, whereas Erk activationrequired higher concentrations of GGF2. The differential activation ofthe two pathways at low concentrations, as a result, generated a narrowwindow of doses (0.003 to 0.01 nM, boxed in FIG. 4B) in which Akt wasthe predominant pathway activated in response to GGF2. Next, the effectof various doses of GGF2 on myelination was determined in the co-culturesystem. At the low concentration window, GGF2 elicited adosage-dependent promyelinating effect: 1.5-, 2.3-, 2.2- and 2.8-foldincrease in myelination compared to the control cultures at 0.0005,0.001, 0.003 and 0.01 nM of GGF2, respectively (FIG. 4C). As theconcentration increased further, GGF2 began to inhibit myelination,coinciding with the appearance of Erk activation. The promyelinatingeffect of GGF2 was also demonstrated in CRD-Nrg1^(+/−) co-cultures inwhich low doses of GGF2 rescued the myelination defect on the mutantaxons (FIG. 4D).

Soluble Nrg1 can both promote and inhibit myelination: binary choicedetermined by the concentration: In the peripheral nervous system (PNS),GGF2 has been regarded as an Nrg1 isoform associated with the Schwanncell injury response. Ectopic in vivo expression of GGF-033 inmyelinating Schwann cells stimulates cell proliferation and inducesdemyelination (Huijbregts et al. J Neurosci 2003; 23:7269-80). Moreover,addition of high concentrations of GGF2 (e.g., those exceeding 0.25 nMGGF2) to Schwann cell-DRG neuron co-cultures has been shown to inhibitmyelination (Zanazzi et al. J Cell Biol 2001; 152:1289-99). Thus, anunexpected result of the present study was the discovery that at lowconcentration, GGF2 exhibits myelination promoting effects. Thepromyelinating effect was, however, limited to a low concentration rangeand an increase in GGF2 concentration from this range results ininhibition of myelination as described previously. This is an intriguingfinding as it demonstrates that soluble GGF2 can elicit two contrastingbiological functions under the same cellular context solely based on theamount presented to the cell. It also suggests that threshold levels ofGGF2 determine the promyelinating and inhibitory function duringmyelination. As demonstrated for the first time in the present study,this can be explained by concentration-dependent differential activationof the receptor downstream signaling effectors. More specifically, thepresent data show that the promyelinating function of GGF2 is observedat concentrations that preferentially activate Akt, while the transitioninto the inhibitory role at higher concentrations coincides with theappearance of Erk activation despite the continuous increase in thelevel active Akt. This result also supports the previous notion that thebalance between 1³13-kinase and Ras/Raf/Erk activation is crucial indetermining the state of myelination in Schwann cells (Ogata et al. JNeurosci 2004; 24:6724-32). The present findings, however, providedirect evidence that activation of the Ras/Raf/Erk pathway functions asa negative regulator of myelination.

The inhibitory function Nrg1 on myelination is mediated through Erk/Mek1activation: The inhibitory role of Ras/Raf/Erk pathway on myelinationhas been suggested previously by studies wherein expression ofconstitutively active Mek1 in Schwalm cells blocks forskolin-inducedmyelin gene expression, whereas dominant-negative Ras blocks myelin genedown-regulation induced by Nrg1. Its direct effect on myelination,however, has not been elucidated prior to the instant results. Asdemonstrated herein, GGF2, when used above a threshold concentration,inhibits myelination in the co-cultures. The present inventors showherein that inhibition of Mek1/Erk1 activation restored myelination inGGF2-treated co-cultures, demonstrating that the inhibitory role of GGF2was mediated through its Ras/Raf/Erk1 activation. The mechanism by whichMek1/Erk activation inhibits myelination is unclear. A possiblemechanism includes suppression of myelin gene expression as describedpreviously. Supportive of this suggestion, the present data revealedthat an increase in myelination in U0126 treated cultures wasaccompanied by an increase in P0 expression. It is also possible thatthe Mek1/Erk pathway might modulate expression of transcription factorsinvolved in myelination or Schwann cell differentiation. Recently it hasbeen shown that ectopic expression of c-Jun in Schwann cells inhibitsmyelination, thus suggesting that c-Jun functions as a negativeregulator of the myelin program. The present findings are consistentwith this conclusion since the present inventors determined that GGF2treatment that inhibits myelination is accompanied by c-Jun inductionand furthermore, inhibition of the Nrg1-induced Mek1/Erk1 activityblocks c-Jun expression. This result suggests that the inhibitoryfunction of Mek1/Erk1 on myelination is in part mediated throughinduction of c-Jun.

Another interesting finding of the present study is the presence of anintrinsic Mek1/Erk-dependent signal in the co-cultures that serves as anegative regulator of myelination. This was shown in an experiment inwhich treatment of normal myelinating co-cultures with U0126 promotedmyelination. The nature of the signal that contributes to the Mek1/Erk1activity during myelination is presently unknown, although it is likelyto be axonal in origin, independent of the axonal CRD-Nrg1 Possiblecandidates are type I and II Ig-Nrg1 that are expressed by the PNSneurons and later released from the axonal membrane by proteolyticcleavage. Another possible Mek1/Erks activator is FGF-2, which isexpressed in PNS neurons and the receptor for which is expressed onSchwann cells. Treatment with FGF-2 down-regulates myelin geneexpression and inhibits myelination in vitro. Loss of FGF-2 expressionresults in an increase in the number of myelinated axons during sciaticnerve regeneration. Peripheral neurons also express PDGF and IGF, withthe corresponding receptor tyrosine kinases expressed on the associatedSchwann cells. It will be of a great interest to assess the regulatoryrole of these growth factors during myelination of the PNS.

Therapeutic use of GGF2: Experimental transplantation has providedoverwhelming proof for the potential of repairing damaged nerves bytransplantation of myelinating glial cells. Schwann cells are goodcandidates for such therapy as they are easily expanded in culture andoffer the possibility of autologous transplantation to promoteremyelination and restoration of nerve conduction at the demyelinatedlesions not only in the PNS but also in the CNS. Remyelination bySchwann cells on adult regenerating axons, however, is often incompleteresulting in formation of thinner myelin sheath and shorter internodecompared to normal nerves. Therefore, the present demonstration of thepromyelinating function of GGF2 and the ability to avoid inadvertenthindrance of myelination due to GGF2 dose levels is significant as itprovides a therapeutic strategy for the treatment of demyelinatingdiseases as well as for rebuilding myelin following nerve injury.

Other Embodiments: All publications and patent applications mentioned inthis specification are herein incorporated by reference to the sameextent as if each independent publication or patent application wasspecifically and individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure come within known or customary practice within theart to which the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of theappended claims.

1. A method for avoiding inhibition of Schwann cell myelinationfollowing administration of glial growth factor 2 (GGF2) in a subject,said method comprising: providing a subject in need of neuronmyelination; providing GGF2 in a pharmaceutically acceptable carrier;administering the GGF2 to the subject; and, determining that the amountof GGF2 is less than the amount that inhibits Schwann cell myelination.2. The method of claim 1, wherein the GGF2 is administeredintravenously, intrathecally, or topically.
 3. The method of claim 1,wherein the determining step comprises administering less than a maximumnormative value of GGF2.
 4. The method of claim 3, wherein the a maximumnormative value of GGF2 is a plasma level of about 0.01 nM GGF2.
 5. Themethod of claim 3, wherein the a maximum normative value of GGF2 isabout 500 ng of GGF2 per kg of body weight.
 6. The method of claim 1,wherein the determining step comprises determining levels of c-Junfollowing the administering step.
 7. The method of claim 6, wherein ifc-Jun levels are increased, further comprising a step of: administeringa reduced amount of GGF2 to the subject relative to amount administeredinitially.
 8. The method of claim 6, wherein c-Jun levels are determinedin a fluid selected from the group consisting of: intracellular fluid,blood plasma, blood serum and cerebrospinal fluid.
 9. The method ofclaim 1, wherein the determining step comprises determining the amountof GGF2 relative to the amount of GGF2 that induces Mek1/Erk pathwayactivation.
 10. The method of claim 9, wherein determining stepcomprises determining that the amount of GGF2 is below the amount thatinduces Mek1/Erk pathway activation by detecting phosphorylated Erk,wherein the threshold level associated with inhibition of Schwann cellmyelination is an amount of GGF2 sufficient to activate Mek1/Erkpathways.
 11. The method of claim 9, wherein the determining stepfurther comprises determining that the amount of GGF2 is sufficient forPI3-kinase pathway.
 12. The method of claim 11 wherein PI3-kinasepathway activation is assessed by detecting phosphorylated Akt.
 13. Themethod claim 9, wherein when Mek 1/Erk pathway activation is induced,further comprising a step of: administering an amount of a Mek 1/Erkpathway inhibitor to the patient in an amount sufficient to inhibit theMek 1/Erk pathway. 14.-23. (canceled)
 24. A method for determining if anamount of GGF2 is a therapeutically effective amount for promotingmyelination, the method comprising: providing a subject receiving GGF2therapy; and measuring c-Jun protein levels in the subject, whereby anincrease in c-Jun relative to baseline c-Jun levels indicates that theamount of GGF2 is near a maximum threshold of therapeutic efficacy forpromoting myelination.
 25. The method of claim 24, further comprisingdecreasing the amount of GGF2 administered to the subject if c-Junlevels are increased relative to baseline c-Jun levels.
 26. The methodof claim 24, further comprising maintaining the amount of GGF2administered to the subject if c-Jun levels remain at the baseline c-Junlevels.
 27. A pharmaceutical composition comprising GGF2 or an EGFLdomain and a Mek1/Erk pathway inhibitor.
 28. A pharmaceuticalcomposition of claim 27 for use in the treatment of a patient afflictedwith a disease or disorder associated with reduced levels ofmyelination.